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Gold-based anticancer derivatives of oligopeptides: synthesis, characterization, delivery strategies and biological activity
Full text linkAt present, cisplatin (cis-diamminodichloroplatinum(II)) is one of the most largely employed anticancer drugs, used especially for the treatment of testicular cancer and, in combination with other drugs, of ovarian, small cell lung, bladder, brain, and breast tumors. Anyway, despite its high effectiveness, it exhibits severe drawbacks, as normal tissue toxicity (in particular, nephrotoxicity) and intrinsic or acquired resistance to the treatment. To overcome these problems, new Pt- and other metal-based complexes were designed and tested as anticancer drugs. In particular, since gold-based complexes were linked for centuries to the treatment of rheumatoid arthritis (due to their antiinflammatory and immunosuppressive properties), they were also tested for their antiproliferative properties..
In the last decade in our research group, some Au(III)-dithiocarbamato derivatives were synthesized, characterized and tested, showing outstanding antiproliferative activity on human cancer cells.
Starting from the promising results obtained both in vitro and in vivo for the “first generation" compounds of formula [AuIIIX2(dtc-L)] (X= Cl, Br; dtc=dithiocarbamato; L= dimethylamine, pyrrolidine, N-methyl glycine (Sar) esters), a "second generation" of complexes was designed to improve bioavailability, selectivity and cellular uptake. In particular, the dithiocarbamato moiety was functionalized with different oligopeptides of general formula [AuIIIX2(dtc-Sar/Aib-(AA)n)] (AA= different aminoacids), to be recognized and uptaken by specific peptide transporters called PEPTs (PEPT1 and PEPT2), predominantly present in epithelial cells of small intestine, mammary glands, lung, choroid plexus and kidney, and overexpressed in some tumor types. A unique feature of these transporters is their capability for sequence-independent transport of most possible di- and tripeptides, thus representing excellent targets for the delivery of pharmacologically-active peptidomimetics such as ß-lactam antibiotics and some angiotensin converting enzyme (ACE) inhibitors. Interestingly, these complexes demonstrate high capability to strongly inhibit tumor cells growth by exploiting a mechanism of action different from the clinically-established platinum drugs, overcoming drawbacks without any observed cross-resistance.
In this PhD thesis work, new Au(III)-peptidodithiocarbamato complexes were synthesized and fully characterized, mainly by means of elemental and X-ray analysis, mono- and bidimensional NMR, FT-IR and UV-Vis spectroscopy.
The water-insoluble compounds, previously dissolved in DMSO, showed high reactivity in aqueous conditions, generally undergoing hydrolysis of the bromido ligands which leads to the formation of the water insoluble dihydroxo derivative. Nevertheless, when analyzing more complex environments, as cell culture medium, a stabilizing effect seemed to be exerted by the components of the medium itself (e.g., proteins, organic molecules), as confirmed by interaction studies with the serum carrier protein BSA (Bovine Serum Albumin).
The antiproliferative activity of the complexes was tested on different human cancer cell lines, representative of different cancer types (e.g. lung, ovary, breast), showing promising cytotoxicproperties. Furthermore, their antitumor activity was confirmed in vivo on breast MDA-MB-231 human xenografts, while their toxicological profile, preliminary evaluated ex vivo, was similar to the "first generation" compounds.
To overcome the solubility problem in aqueous conditions and to avoid the intrinsic toxicity of DMSO (normally used as primary solvent), new solubilizing methods were explored. In particular, micelles based on Pluronic F127 and liposomes based on DPPC phospholipids were prepared, characterized and tested in vitro. These systems were proved to stabilize the compounds in physiological conditions, without affecting their antiproliferative activities. Moreover, micelles functionalized with CCK8 octapeptide were tested as targeting moieties to be selectively delivered to cancerous cells overexpressing the CCK-receptors. In this case, the GI50 values of the encapsulated compounds were improved of one order of magnitude.
To achieve more information about their mechanism of action, the interaction in vitro with some purified enzymes was performed, confirming that the complexes inhibit all the three enzymatic functions of the proteasome, together with the activity of PARP-1 enzyme.
More detailed studies, taking into account the kinetics of gold uptake by cells and the real-time inhibition of cell growth and adhesion, suggested that the complexes probably exert their cytotoxic effect on cells in the 4-5 h after administration, acting probably at the membrane level.
All together these results highlight the promising properties of the complexes as anticancer agents, and put the basis for the pursuance of the preclinical characterization, including deeper studies of their pharmacological properties and biological activity.
I wish to aknowledge my supervisor Prof. Dolores Fregona and all her Research Group.
A particular thanks goes to Prof. Dalla Via, Prof. Formaggio, Prof. Di Noto.
I want also to acknowledge Prof. Angela Casini for the ospitality in her Research Group at the Research Institute of Pharmacy of the University of Groningen (The Netherlands).
A special thank goes to Fondazione CARIPARO, for funding this PhD project.
Finally, I want to thank the Doctoral School of Molecular Sciences, its Coordinator Prof. Antonino Polimeno and the members of my three-year committee Prof. Formaggio, Prof. Badocco and Prof. Zagotto
Insights into the reactivity of gold(III)-dithiocarbamato anticancer agents toward model biomolecules from multinuclear NMR spectroscopy
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Chiral gold(III)-dithiocarbamato peptidomimetics for the anticancer chemotherapy
No full textPeptide-based anticancer drugs are attracting much interest since they can target PEPT1
and PEPT2, membrane proteins able to transfer di- and tripeptides through the cell membrane.
As these proteins seem to be overexpressed in some types of tumors, [1] we recently modified
our promising gold(III)-dithiocarbamato (dtc) derivatives [2,3] with peptide ligands.[4,5]
Interestingly, the complexes of the type [AuIIIX2(dtc-Sar-AA-O(t-Bu))] (X = Cl, Br; AA =
Gly, Aib (α-aminoisobutyric acid)) exhibited higher anticancer activity than cisplatin, together
with being not toxic towards healthy cells. Peptide ligands longer than a dipeptide did not
improve the biological properties of our compounds.[5]
The three amino acids employed so far (Sar, Gly and Aib) are achiral. In addition, Sar
and Aib are not contained in proteins, although they are naturally occurring. Therefore, we
decided to investigate the role of chirality and of the protein amino acid residues on the
biological activity of our leading compounds. In this communication, we report on the
synthesis and characterization of a new series of complexes: [AuIIIX2(dtc-l-Pro-Aib-O(t-Bu))],
[AuIIIX2(dtc-d-Pro-Aib-O(t-Bu))], [AuIIIX2(dtc-Sar-l-Ala-O(t-Bu))] and [AuIIIX2(dtc-Sar-d-
Ala-O(t-Bu))]. We also prepared [AuIIIX2(dtc-Sar-Aib-OTEG)], where the introduction of
TEG (triethylenglycol) is aimed at improving the water solubility of the complex. By
exploiting the Wallach rule (racemates pack better), we managed to grow single crystals of
[AuIIIBr2(dtc-l,d-Pro-Aib-OtBu) and solve the X-ray crystal structure. Anticancer and toxicity
tests of all newly synthesized compounds are currently in progress
Perspective gold(III)-dithiocarbamato anticancer therapeutics: learning from the past, moving to the future.
No full textThe unquestionable therapeutic success of the anticancer drug cisplatin and its second and third generation analogues has triggered, during the past fifty years, the development of several metal-based potential chemotherapeutic agents, most of which have unfortunately failed to enter clinical trials. In this context, since the early late 1990s, the Biomedicinal Chemistry Research Group at the University of Padova (Italy) has been making quite an effort to design a number of metal-dithiocarbamato derivatives that were expected, at least in principle, to resemble the main features of cisplatin together with higher activity, improved selectivity and bioavailability, and reduced side-effects. Among all, some selected gold(III) complexes have been showing outstanding in vitro and in vivo antitumor properties and negligible (or even no) acute and renal toxicity, compared to the reference clinically-established platinum drugs.
Starting from the rationale behind such investigations, results achieved to date are here summarized, focusing on the in-depth mechanistic studies that have been providing insights into the mechanism of action of this class of metal compounds. New prospects opened up by these anticancer agents, including the latest development of “second generation” gold-based peptidomimetics for the targeted chemotherapy, are also illustrated and discussed
Beyond Platinums: Gold Complexes as Anticancer Agents
Full text linkThe accidental discovery of the anticancer properties of cisplatin in the mid-1960s triggered the development of alternative platinum-based drugs. However, the platinum-based treatment of tumor diseases is massively hampered by severe side-effects and development of resistance. Sulfur-containing biomolecules play a significant role in platinum anticancer chemotherapy because of their high affinity to the platinum(II) ion. Sulfur is involved in the entire metabolic processing of platinum drugs. Strong and irreversible binding of cisplatin to intracellular thiolato ligands is considered a major step of inactivation, and reactions with sulfur donors in proteins are believed to affect enzymatic processes. Consequently, the development of novel metal-based compounds with a pharmacological profile different from that of clinically-established platinum drugs is a major goal of modern medicinal chemistry and drug design. Among the non-platinum antitumor agents, gold(III) complexes have recently gained increasing attention due to their strong tumor cell growth-inhibiting effects, generally achieved by exploiting non-cisplatin-like mechanisms of action. The real breakthrough is not simply the use of gold compounds to treat cancer, but the rational design of gold-based drugs which may be very effective, non-toxic and potentially selective towards cancer cells, their potential impact relying on the possible site-specific delivery in localized cancer, thus strongly improving cellular uptake and minimizing unwanted side-effects. Cancer cells are known to overexpress specific proteins and receptors needed for tumor growth. Among them, two integral plasma membrane proteins mediate the cellular uptake of di- and tripeptides and peptide-like drugs. They are present predominantly in epithelial cells of the small intestine, bile duct, mammary glands, lung, choroid plexus, and kidney but are also localized in other tissues and are up-regulated in some types of tumors. Accordingly, we have been designing gold(III)-peptide dithiocarbamato derivatives which combine both the antitumor properties and reduced side-effects of the previously reported gold(III) analogues with enhanced bioavailability and tumor selectivity achieved by exploiting peptide transporters. Our compounds showed interesting cytotoxic properties towards a number of cancer cell lines in vitro and in vivo on xenograft models, together with negligible organ and acute toxicity. With respect to their mechanisms of action, we identified mitochondria and proteasome as major in vitro and in vivo targets. These results allowed the filing of an international patent for the use of gold(III) peptidomimetics in cancer chemotherapy, as well as providing a solid starting point for them to enter phase I clinical trials in a few months
t-Butylsarcosinedithiocarbamato gold(III)-based anticancer agents: design, in vitro biological evaluation and interaction with model biomolecules
No full textAs a further extension of our research work focusing on the development of gold(III)-dithiocarbamato derivatives as potential anticancer agents, the new complexes [AuIIIX2(dtc-Sar-O(t-Bu))] (X = Cl (1)/Br (2)) are here reported. The compounds were characterized by means of FT-IR, ESI mass, and mono- and multidimensional NMR spectroscopy. In order to get further insights into the possible behavior under physiological conditions, their affinity toward selected model biomolecules was spectroscopically investigated in detail. Remarkably, they seem to react very slowly with isolated dAMP (but not dGMP), forming a species identified as [AuIII(dtc-Sar-O(t-Bu))(dAMP-N1,C6NH)]+. In presence of GSH they undergo sudden reduction to the gold(I) counterpart [AuIII2(dtc-Sar-O(t-Bu))2], whereas only secondary interactions seem to occur when reacted with BSA. According to in vitro cytotoxicity studies, both complexes turned out to be highly effective toward all the human tumor cell lines evaluated (HeLa, L540 and U937), reporting IC50 values lower than cisplatin. Apoptosis was proved a major cell death mechanism and, accordingly, DNA fragmentation was observed. Remarkably, cell cycle progression was negligibly affected, thus supporting the hypothesis a different mechanism of action from clinically-established platinum-based drugs
Selective delivery of chemotherapeutics into tumor cells by use of targeting peptides
No full textGold(III)-dithiocarbamato complexes have recently gained increasing attention as
potential anticancer agents because of their strong tumor cell growth-inhibitory effects,
generally achieved by exploiting non-cisplatin-like pharmacodynamic and pharmacokinetic
properties and mechanisms of action. In particular, gold(III) complexes share with
platinum(II) derivatives some key chemical features, such as the preference for square-planar
geometry and the typical d8 electronic configuration, making them attractive for testing as
antineoplastic drugs. Moreover they have been successfully used as chemoprotectants to
modulate cisplatin nephrotoxicity without decreasing its antitumor activity. In this context, we
have previously reported on some gold(III)-dithiocarbamato derivatives of the type
[AuIIIX2(dtc)] (X = Cl, Br; dtc = various dithiocarbamates) that reproduce very closely the
main features of cisplatin. These gold(III) compounds were shown to exert outstanding in
vitro cytotoxicity, even toward human tumor cell lines intrinsically resistant to cisplatin, and
no cross-resistance to the reference platinum drug.[1]
In order to obtain selective drug targeting to cancerous cells, peptide conjugates of Aucomplexes
have been synthetized by combining different synthetic approaches (solid phase
synthesis and in solution methods). The bioactive peptide selected was the octapeptide CCK8,
which corresponds to the eight-residue C-terminal end of the cholecystokinin peptide
hormone, and has been widely studied for its high binding affinity, in the nanomolar range,
towards the two cholecystokinin receptors, CCK1-R and CCK2-R.[2] An alternative strategy
to deliver anticancer drug selectively to tumor cells was the loading of the Au-complexes in
supramolecular aggregates (micelles or liposomes) externally functionalized by peptides.[3
Ruthenium(II/III) Complexes of S-donor Ligands: Synthesis, Characterisation and in vitro Cytotoxicity Activity toward Lung Cancer Cells
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